In the field of immunoassaying, reacting species are often very dilute, which results in assays that require an extended incubation time to provide an observable result and which are insensitive.
For example, for the assay of some serum constituents such as traces of hormones (e.g., ACTH), the concentration is so low that the reaction with an approximately stoichiometric equivalent of specific antibody may take more than 24 hours to complete. (See, e.g., Radioimmunoassay & Related Techniques Methodology & Clinical Application by Thorell & Larson, published by C. V. Mosby Co., St. Louis, 1978, pp. 144, 186, 198, 200).
If the concentration of antibody and antigen were to be increased by 300 times each, the bimolecular reaction rate between them will be speeded by 300.times.300=90,000 times, which results in reducing a 24 hour reaction rate to less than 1 second.
When the label used in an immunoassay is a chromophore, fluorophore, spin label, etc., the sensitivity with which it can be assayed is directly proportional to the concentration of the label in the final assay volume (when the detector system has been scaled to that volume). Therefore, these methods, which concentrate the immunoreactants to speed the reaction, also provide a substantial increase in assay sensitivity.
Despite the fact that preconcentrating has been recognized as desirable, to date there is no convenient means of accomplishing this result. Reactants can be preconcentrated by centrifugation, but this technique is not entirely satisfactory. In the first instance, the preconcentrated reactants often must be partly rediluted when they are removed from the centrifuge for reaction purposes. Secondly, this procedure usually requires expensive equipment. Thirdly, the procedure is inordinately time consuming. This is especially so for species of low molecular weight such as small antigens like angiotensin and haptens.
The invention achieves preconcentration of at least one reactant of the reaction in the very medium in which the reaction is accomplished. This eliminates the aforementioned drawback of redilution. In addition, the invention causes the reactants to preconcentrate at concentrations many times greater than generally achievable in prior art methods as applied to immunoassays. Furthermore, the invention seeks to achieve all these objectives at low cost, and in a rapid manner.
An assaying technique of the prior art features the reaction of immunoreactants, antigen (or hapten) on one hand, and antibody on the other, within a localized zone of a gel medium. The test substance is caused to migrate by electrophoresis through the gel into reactive contact with the immobilized reactant. After equilibration takes place, the unreacted or unbound substances are separated by further electrophoresis away from the immobilized reactant. Such a system as described above, may be seen with reference to U.S. Pat. No. 3,966,897. This invention, however, does not teach how the reactants may be preconcentrated within the gel reaction medium.
Another technique which has been known to concentrate constituents of a fluid sample within a medium is known as discelectrophoresis. This method was invented by L. Ornstein, one of the present inventors, and is described in U.S. Pat. No. 3,384,564, issued May 21, 1968. This technique features developing a discontinuous electric field across a gel medium. Different constituents of a fluid sample with the same sign charge will initially migrate through the gel at different speeds. Each constituent will rapidly concentrate into a narrow band, the process terminating in a steady state with all the same sign of charge constituents migrating at the same speed with a distinct order in the gel.
While this technique has been used to separate and concentrate components of a sample, it never has been used or suggested as a method for bringing together two immunological species at high concentration for performing a controlled reaction.
The present invention is generally distinguished from the prior immunoassay art by means of its selective sieving of the constituents of a reaction in order to concentrate them. In the prior immunoassay art, as molecules of the reactants migrate through a medium in an electrophoretic field, they also tend to randomly diffuse in every direction. Consequently, a dilute substance traveling through a liquid or gel medium will tend to further dilute. This is so, because some reactant molecules will be diffusing backwardly even while the overall mass of substance is moving forwardly through the medium in an electric field. In the prior art, the ultimate potential for achieving extremely rapid reaction rates is never fully realized. This is because the reactants are not brought together in concentrated form.
In the invention, a gel or non-convecting medium is selected to eliminate non-random convective migration of the reactant molecules. One of the reactant species is caused to migrate through the medium towards a concentration or barrier zone which is permeable to small ions but impermeable to the reactant species. This concentration or barrier zone does not allow further migration of the arriving reactants. The continuing electrophoretic force will then cause the molecules of the substance to concentrate in a narrow zone very close to the barrier, such that the reactants will become very concentrated within a very small volume. If the other reactant species is already concentrated and disposed within the concentration zone, then the arrival and concentration of the complementing reactant wil initiate a very rapid reaction. In another embodiment, the second reactive species can be added to the system after the first has concentrated. It too will migrate towards the barrier and concentrate in the same region as the first. In the case of some immunoreactions, it may be required that subsequent to the reaction, bound and unbound reactants be separated. This may be achieved, for example, by reversal of the direction of the electrophoretic force. In this case, the reacted species can be arranged to be immobilized in the medium, because its size has been increased as a result of the reaction and it cannot migrate in the medium. The molecules of the unreacted species can, however, move out of the reaction zone as rapidly as they moved in. The determination of the unknown reactant in the sample can then be measured within the medium by known procedures.